The Molecular Mechanism Of Transcript Factor TWIST1 Affecting On DAC Resistance In MDS/AML

Myelodysplastic syndromes(MDS)are a group of clonal,heterogeneous hematopoietic stem cell diseases with a differentiation disorder caused by malignant changes at the level of totipotent stem cells.They have high risk of transforming into acute myeloid leukemia(AML).AML is a malignant hematological tumor that originates in hematopoietic stem/progenitor cells and is characterized by abnormal proliferation of myeloid precursor cells in bone marrow and peripheral blood.In recent years,increasing incidence of MDS/AML has seriously threatened human life and health.With the development of epigenetics research,more and more studies have found that abnormal DNA methylation plays an important role in the occurrence and development of MDS/AML.Therefore,demethylation drugs(such as decitabine and azacitidine)are widely used in the treatment of various subtypes of MDS/AML.However,there are still some patients with obvious drug resistance having no significant improvement or showing recurrence after receiving demethylation treatment.Therefore,understanding the mechanism of resistance to demethylation in patients with MDS/AML is essential.TWIST1 is a transcription factor with a basic helix-loop helix structure which can recognize and bind E-box sequences to regulate target gene expression.Studies have confirmed that the expression of TWIST1 in hematopoietic stem cells of high-risk MDS patients was significantly increased and TWIST1 has been proven to be potentially carcinogenic.In previous experiments,we found that the genome methylation level was increased and gene transcription was suppressed in CD34~+cells that overexpressing TWIST1.Therefore,it was speculated that the expression of TWIST1 in MDS/AML was closely related to DNA methylation.It was found from clinical samples that TWIST1 levels of mononuclear cells were higher in DAC non-responsive(DAC-NR)group than in DAC responsive(DAC-R)group,suggestting that TWIST1 was involved in regulating decitabine resistance.Therefore,in this study we systematically elucidated the molecular mechanism of TWIST1 involved in regulating DNA methylation and resistance to decitabine in myeloid malignant clone cells through cellular and murin modles.The main conclusions are as follows:1.Analysis of the correlation between TWIST1 expression and decitabine resistance:TWIST1 levels of mononuclear cells were higher in DAC-NR group than in DAC-R group.Follow-up experiments showed that global methylation levels were significantly higher in DAC-NR than in DAC-R,and DNMT3a expression,which can initiate de novo methylation,was higher in DAC-NR than in DAC-R Consistent with this clinical data,TWIST1overexpression in CD34+cells from cord blood was associated with increased DNA methylation and transcriptome suppression,whereas silencing of TWIST1 expression in these cells reduced DNA methylation.Meanwhile,AC-resistant KG1a cells(KG1a-DAC-R),generated by continuous culture and treatment with DAC at gradually increasing concentrations,showed enhanced DAC resistance,significantly higher TWIST1 expression,and increased global methylation levels.To clarify the role of TWIST1 in DAC-mediated cell proliferation,TWIST1 was overexpressed in KG1a(KG1a-TWIST1)and was stably knocked down in SKM1 and OCI-AML3.Cell proliferation was higher in KG1a-TWIST1than in parental KG1a,while inhibition of TWIST1 in SKM1 and OCI-AML3 resulted in reduced cell proliferation.Results of xenotransplantation murine model showed the engraftment of KG1a-TWIST1 cells was significantly higher in peripheral blood,spleen and bone marrow than that of KG1a under decitabine treatment.2.Identification of TWIST1 and DNMT3a interactions and binding sites:Analysis of tandem affinity mass spectrometry revealed that multiple proteins involved in genomic methylation and chromatin modification interacted with TWIST1,including the methyltransferase DNMT3a;Co-immunoprecipitation detection showed the direct binding of TWIST1 and DNMT3a in KG1a-TWIST1,SKM1 and OCI-AML3.TWIST1 and DNMT3a was co-localized in the nucleus detected by cell immunostaining;Epitope mapping assay was identified that there are four consecutive polypeptide fragments(peptides 19-23,53-57,60-64,66-70)of the TWIST1 protein that could bind to DNMT3a.The peptides 66-70(sequence was LRKIIPTLPSDKLSKIQTLKLAA)were confirmed to inhibit the binding of TWIST1a and DNMT3a by ELISA and co-IP;Further,cells were treated with peptides66-70 could effectively enhance the sensitivity of KG1a-TWIST1 to decitabine;KG1a-TWIST1 was injected into NSG mice through the tail vein to build the xenotransplantation model.Then,decitabine or mixture of decitabine and peptides 66-70 were injected for 6times,respectively.Compared with NSG mice only treated with decitabine,the engraft of KG1a-TWIST1 cells in peripheral blood of mice treated with peptides 66-70 and decitabine significantly reduced,consistent with in vitro results.These data indicated peptides 66-70could enhance the sensitivity of myeloid malignant clone cells to decitabine by inhibiting the binding of TWIST1 to DNMT3a.3.TWIST1/DNMT3a complex regulated downstream target gene methylation:Methylation chip results showed that the methylation level of genes whose promoter region contained E-box motif changed significantly in overexpressed TWIST1 cells.The methylation levels of the cell cycle kinase-dependent inhibitor CDKN1A and CDKN1C were significantly increased,and the specific methylation PCR results were consistent with the methylation chip results;Our experiments revealed suppression of CDKN1A/C m RNA levels in KG1a-TWIST1.We observed significant increase of m RNA expression and reduction of methylation level of these two genes in KG1a relative to KG1a-TWIST1 following DAC treatment.Flow cytometric analysis showed that DAC treatment resulted in G0/G1 phase arrest in KG1a,and to a lesser degree in KG1a-TWIST1;Results of CD34+cells that overexpression of TWIST1 indicate that TWIST1 recruits DNMT3a to methylate promoter regions of CDKN1A/C,resulting in inhibition of CDKN1A/C expression and consequent reversal of G0/G1 arrest.In MDS/AML patients,methylation levels of CDKN1A/C promoter areas were higher in DAC-NR than in DAC-R group,and CDKN1A/C expression was lower in DAC-NR.4.Effect of O-GlcNAc glycosylation on expression and function of TWIST1:Analysis of clinical samples showed that OGT expression of mononuclear cells in DAC-NR was significantly higher than that of mononuclear cells in DAC-R group.Flow cytometry results showed that KG1a-TWIST1 cells were more sensitive to decitabine under OGT inhibitor(OSMI-1)treatment;Co-immunoprecipitation showed TWIST1 has O-GlcNAc modification;Further,OSMI-1 could inhibit the binding of TWIST1 and DNMT3a.Expression of TWIST1 protein decreased with increased concentration of OSMI-1 and with increased time of OSMI-1;Treatment of OSMI-1 caused increased ubiquitination of TWIST1 and its degradation via proteasomes pathway,indicating O-GlcNAc glycosylation could stablize TWIST1;We also found TWIST1 could bind with OGT promoter region and activate OGT transcription by chromatin coprecipitation and dual luciferase reporter gene experiments.